Sympathetic and parasympathetic innervation in hepatocellular carcinoma.

Autonomic nervous system plays an important role in the development of multiple cancers via regulating cancer cell proliferation, differentiation, apoptosis, migration and invasion. However, no detailed studies have been performed to study the role of autonomic nerve fibers in hepatocellular carcinoma (HCC) as well as its correlation with the progression of HCC. Here, we examined the distribution of the autonomic nerve fibers and analyzed the correlation between autonomic nerve fibers and the pathological characteristics of HCC patients. The transcriptional expression of adrenergic and cholinergic receptors was evaluated in both hepatoma cell lines and primary hepatoma cells. In addition, we summarized the function of receptors for neurotransmitters in different cancers recently reported. Our findings indicate that tissue of liver cancer is innervated by both sympathetic and parasympathetic nerves and the density of the nerve fibers is associated with patients' poor prognosis. Additionally, we report that adrenergic receptors ß2 and cholinergic receptors α7, M1 and M3 are high expressed in both hepatoma cell lines and primary hepatoma cells, indicating these receptors may play essential roles in the regulation of autonomic nervous system triggered HCC.

BMP-2 promotes chondrogenesis of rat adipose-derived stem cells by using a lentiviral system.

Osteoporosis poses a major public health threat in aging societies. Adipose-derived stem cells (ADSCs) are multipotent adult stem cells that have the ability to yield mesenchymal stem cells, and have the potential to undergo osteogenesis and bone regeneration. Bone morphogenetic proteins (BMPs) have been demonstrated to upregulate bone gene expression after mechanical injury and to improve bone injury repair. This study aimed to produce BMP-2 expression in ADSCs by using lentiviral vectors. Subcutaneous adipose tissue from 4-week-old male Sprague-Dawley rats was used. Oil red O staining was used to detect adipocyte formation from ADSCs. Induction of ADSC osteogenesis was confirmed with Alizarin red S staining. The recombinant lenti-hBMP-2/neo was constructed to infect ADSCs, BMP-2 expression was measured by immunoblotting analysis, and cellular alkaline phosphatase levels were examined. We found that >70% of ADSC cells could be induced to differentiate into osteocytes or adipocytes. Under osteogenic induction, ADSCs showed increased intracellular calcium deposition, the formation of calcium tubercles, and the disappearance of cellular structures in calcium tubercles. After infection of ADSCs by lenti-hBMP-2/neo, BMP-2 was expressed after doxycycline induction. We, thus, conclude that ADSCs maintain vigorous growth ex vivo and possess stem cell-like properties. When infected with lenti-hBMP-2/neo, ADSCs can be induced to promote BMP-2 expression.

Observation of photorefractive surface waves in self-defocusing LiNbO3:Fe crystal.

Photorefractive (PR) surface waves (SWs) in self-defocusing LiNbO(3):Fe are studied theoretically and experimentally. We demonstrate that SWs can also be formed in a self-defocusing nonlinear medium and that the nonlocal nonlinearity (such as the diffusion component of PR nonlinearity in this Letter) is the essential cause. The forming process of PR SWs with a self-deflection course of light beams has been observed. The results indicate the possibility of concentrating light energy in self-defocusing media, taking advantage of SWs.

Tissue inhibitor of metalloproteinase-1 decreased chemosensitivity of MDA-435 breast cancer cells to chemotherapeutic drugs through the PI3K/AKT/NF-кB pathway.

TIMP-1 is well known to be capable of inhibiting apoptosis. Elevated levels of TIMP-1 in tumor tissue have been shown to be strongly associated with a poor response to chemotherapy. In this study, using conventional cytotoxic drugs commonly used in the treatment of breast cancer, we investigated how TIMP-1 influenced the efficacy using breast cell lines. Our data demonstrated that overexpression of TIMP-1 could significantly decrease the sensitivity of MDA-435 breast cancer cells to epirubicin and paclitaxel. TIMP-1 can potently activate phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-кB) signaling. Furthermore, the TIMP-1-induced attenuation of the effect of epirubicin and paclitaxel was reversed by the PI3K/Akt chemical inhibitor LY294002 and the NF-кB inhibitor pyrrolidine dithiocarbamate (PDTC), showing that the PI3K/Akt and NF-кB signaling pathway was involved in the TIMP-1-induced effect on chemoresistance. Taken together, our results indicate that TIMP-1 decreased chemosensitivity through the PI3K/Akt/NF-кB signal transduction pathway in MDA-435 breast cancer cells.

An investigation of models of the IP3R channel in Xenopus oocyte.

We consider different models of inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) channels in order to fit nuclear membrane patch clamp data of the stationary open probability, mean open time, and mean close time of channels in the Xenopus oocyte. Our results indicate that rather than to treat the tetrameric IP(3)R as four independent and identical subunits, one should assume sequential binding-unbinding processes of Ca(2+) ions and IP(3) messengers. Our simulations also favor the assumption that a channel opens through a conformational transition from a close state to an active state.

Preparation of a respiratory syncytial virus human reference serum for use in the quantitation of neutralization antibody.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants, young children and the elderly. Yet, the development of a vaccine to protect against RSV infection still remains an unmet need. At present, immune responses to experimental vaccines under investigation are usually evaluated by ELISA and/or by neutralization assays against RSV. However, both types of assays are generally performed somewhat differently at different laboratories. An important step towards standardization of serology is the use of a standard human reference serum enabling normalization of results generated within and between laboratories. To fill this need, we prepared and characterized a human reference serum against the A2 strain of respiratory syncytial virus. The serum represents a pool of more than 400 individual human sera obtained from commercial sources. The sera were screened and selected on the basis of individual RSV neutralization titers. A final neutralization titer of 973 (95% C.I., 884-1072) was assigned to the final reference serum pool after it was tested three times in the presence of 10% guinea pig complement and a titer of 286 (95% C.I., 243-337) was assigned to the serum when it was tested in the absence of an exogenous complement source. Sterilely reconstituted lyophilized aliquots of the serum exhibited a stable neutralization titer for at least 1 month at room temperature and at 4 degrees C, as well as after 5 weekly freeze-and-thaw cycles at -20 degrees C. In the lyophilized state, the neutralization titer of the lyophilized reagent was stable for at least 6 months, the last time point tested. Two additional smaller pools of serum with high and medium neutralization titers of 2692 and 575, respectively, were also produced in parallel for use as positive controls and were designated as control sera. The reference serum can be used to normalize neutralization and/or other RSV-specific assay results from different laboratories and the control sera can be used for quality control purposes or as part of a panel to test operator proficiency. Individual lyophilized aliquots of the reference and control sera may be obtained from the US National Institute of Allergy and Infectious Diseases (NIAID) Reference Reagent Repository.

Evaluation of adaptive responses to cisplatin in normal and mutant cell lines with mutations in recombination repair pathways.

Cell lines mutant in specific DNA repair pathways were used to determine if these pathways are involved in adaptive responses. For these studies, the effect of deficiencies in homologous recombination repair (HR) were studied in the parental AA8 and mutant (irs)ISF cell line pair and for deficiencies in the nonhomologous endjoining (NHEJ) pathway in the mouse MEF parental and Ku80 mutant cell line pair. The results showed that the XRCC3 mutation in the HR-deficient mutant inhibited adaptive responses to low doses of cisplatin and radiation. The parental lines showed transient adaptive responses to both low-dose cisplatin and radiation treatment. For the mouse MEF and the Ku80 cells, no adaptive responses were observed in either cell line. However, there was an initial transient sensitization response followed by partial recovery. Thus, it appears that the HR repair system may be involved in the adaptive response to cisplatin and radiation. For the NHEJ repair system the question could not be answered since no adaptive responses were evident in the parental line.

The evaluation of thermal cisplatin sensitization in normal and XP human cells using mild hyperthermia at 40 and 41 degrees C.

The effect of protracted mild hyperthermia treatment at 40 and 41 degrees C given, concurrently with cisplatin, was evaluated in human normal AG1522 and human mutant XPA cells. While mild hyperthermia itself for up to 6 hours showed little to no toxic effects, it did result in significant sensitization of response to cisplatin treatment. Sensitization for the normal and mutant cell line was comparable, indicating that nucleotide excision repair (NER) probably does not have a role in this process. For the 41 degrees C heating, thermotolerance developed and heating times greater than 4 hours resulted in protective effects from cisplatin cytotoxicity. This was not observed for heating at 40 degrees C for up to 6 hours.

Cisplatin sensitization by concurrent mild hyperthermia in parental and mutant cell lines deficient in homologous recombination and non-homologous endjoining repair.

The effect of mild hyperthermia on cisplatin sensitization was examined in two cell line pairs, CHO parental AA8 and irsISF, an XRCC3 mutant (deficient in homologous recombination repair), and mouse parental MEF and knockout Ku80 mutants (deficient in non-homologous endjoining repair). The results showed that mild hyperthermia 40, 41 and 42 degrees C given concurrently with cisplatin treatment caused significant sensitization. The degree of sensitization was comparable for the parental and mutant lines, indicating that these repair pathways were likely not involved in cisplatin thermal sensitization. The shorter concurrent treatments cause a larger sensitization than the longer treatments. The reasons for this are not clear, but thermotolerance may be a factor.

Is DNA polymerase beta important in thermal radiosensitization?

Thermal radiosensitization was tested in a pair of mouse cells (MB+ wild-type and MB-, DNA polymerase beta knockout cells) and in human breast carcinoma cells (MCF7 wild-type and C716 transfected to give elevated DNA polymerase beta expression). Results showed that neither reducing DNA polymerase beta (involved in base excision repair) nor increasing it had any significant effect on thermal radiosensitization. The data indicated that polymerase beta was not involved in thermal radiosensitization, and since hyperthermia is known as a radiation damage repair inhibitor, other repair pathways might be involved and need to be explored.

Comparison of human tumour cell responses to cisplatin and ZD0473 with and without irradiation.

Three pairs of human tumour cell lines, with one line of each pair resistant to cisplatin, were used to compare the effects of cisplatin and ZD0473 on cellular toxicity and radiosensitization. Whilst all three cell line pairs had one line that was resistant to cisplatin, for ZD0473 the lung tumour HTB56cp and cervical carcinoma ME180 cell lines did not express resistance to their HTB56 and SHA counterparts, respectively. Only the ovarian carcinoma line A2780cp showed resistance to ZD0473 compared to its counterpart A2780S. For radiosensitization both cisplatin and ZD0473 show additive and subadditive effects in the ovarian carcinoma lines, and additive and superadditive effects in the cervical carcinoma and lung tumour cell lines. In fact in the lung tumour cell lines ZD0473 appeared to be a more effective radiosensitizer than cisplatin.

Comparison of response to radiation, hyperthermia and cisplatin in parental and polymerase beta knockout cells.

The role of polymerase beta in response to radiation, cisplatin and hyperthermia was examined in a pair of mouse cell lines, comprising a normal parental line and a derivative with polymerase beta knockout. Cell survival was assessed using the colony survival assay. For irradiation, there was no difference in response between the two cell lines. Treatment with cisplatin for 1 h showed a large increase in resistance in the mutant cell line. The results with hyperthermia were more complex. The mutant was more resistant to 45 degrees C heating, but was slightly more heat sensitive than the wild type at 41 degrees C. Thus, in summary, while the knockout of polymerase beta did not alter radiation sensitivity, it did increase resistance to cisplatin and induced resistance to hyperthermia at higher temperatures (45 degrees C).

Thermal radiosensitization and repair inhibition in human melanoma cells: a comparison of survival and DNA double strand breaks.

Human melanoma cells (SK-mel-3) were treated with combinations of radiation and hyperthermia treatment and survival (using the colony forming assay) and DNA double strand breaks (dsb's) (using pulsed field gel electrophoresis) were measured for immediate and delayed plating. The cells were treated in plateau phase, so that delayed plating would result in repair of potentially lethal damage (PLD). Delayed plating showed PLDR for both the survival and the dsb end-point. One hour of heating after irradiation showed a temperature dependent increase in radiosensitization for both the survival endpoint and the dsb endpoint for the temperature range from 42 to 45 degrees C. One hour of heating at 43 degrees C after irradiation resulted in the partial inhibition of PLDR and recovery of dsb's. For heating at 45 degrees C the inhibition of dsb repair was complete. There was good correlation between the survival endpoint and the dsb endpoint for the thermal radiosensitization for both the immediate plating and the PLDR protocols. These data indicate that hyperthermia inhibition of repair of PLD is probably due to the inhibition of dsb rejoining. These correlations were made at the same dose levels for survival and dsb analysis, thus avoiding the potential complications of many earlier studies which used much higher doses for dsb analysis than for survival studies.

Development of a fluorescent focus identification assay using serotype-specific monoclonal antibodies for detection and quantitation of rotaviruses in a tetravalent rotavirus vaccine.

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.

The use of high-resolution solid-state NMR spectroscopy and differential scanning calorimetry to study interactions of anaesthetic steroids with membrane.

We have used a combination of high-resolution solid-state 13C-NMR and DSC (differential scanning calorimetry) to study the distinctively different thermotropic and dynamic properties of the anaesthetic steroid alphaxalone and its inactive congener delta16-alphaxalone in dipalmitoylphosphatidylcholine (DPPC) model membranes. In the solid-state 13C-NMR, the techniques included cross polarization (CP) and/or magic angle spinning (MAS). The observed data revealed the following important results. (a) DSC as a bulk method showed that the active steroid lowers the main phase transition temperature and broadens the pretransition more significantly than the inactive congener. The 13C-CP/MAS experiments allowed us to detect the pretransition temperature in the alphaxalone-containing preparation, which was not discernible in DSC. (b) The chemical shift values varied with temperature, indicating different degrees of trans-gauche isomerization in the lipid acyl chains when the bilayer is in the liquid crystalline phase. (c) Only specific additional peaks appeared in the 13C-CP/MAS spectra when each of the steroids was present in the preparation. delta16-alphaxalone gives rise to more additional peaks than alphaxalone, indicating a different mobility of the corresponding molecular moiety in the phospholipid bilayer environment. (d) The relative intensities of these peaks also confirmed that alphaxalone is fully incorporated in the bilayer, whereas delta16-alphaxalone is only partially so. These results suggest that the differential effects of these two analogues in the membrane may, at least in part, explain the reason for their different biological activities.

Comparison of hyperthermia radiosensitization and DNA polymerase inactivation in human normal and melanoma cell lines of different radiosensitivities.

Two human melanoma cell lines (one radiosensitive, HT144 and one radioresistant, SK Mel-3) and one normal human fibroblast (AG1522) were evaluated for thermal radiosensitization and the thermal enhancement ratios (TERs) were calculated. These were compared with residual polymerase activity to determine if this activity could be used to predict TERs. In all three cell lines, there was a good correlation between TER and residual polymerase alpha or beta activity. Polymerase beta was more sensitive than polymerase alpha as an indicator for TER. There were small cell line-dependent differences (not related to radiosensitivity) among the correlation curves, indicating that for each cell/tumor-type polymerase activity, vs. TER may have to be calibrated.

The conformational properties of the antineoplastic ether lipid 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine.

Thio analogs of platelet activating factor (PAF) are of great interest because they exhibit antineoplastic properties both in vitro and in vivo. In contrast to most known anticancer agents, these lipids appear not to act through the synthesis and function of DNA and, therefore, offer a new avenue of approaching cancer chemotherapy. We have examined the conformational properties of 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine (ET-S-16-OCH3) in organic solvents and in micelles. The conformational analysis was based on a combination of 1D, 2D NMR spectroscopy and molecular graphics. 1H and 13C spin lattice relaxation time (T1) experiments were also performed to study the dynamic properties of this molecule. The picture emerging from these studies is as follows. The alkyl chain of ET-S-16-OCH3 is the most mobile part of the molecule both in CDCl3/CD3OD and in micelles and exists as a mixture of interconverting conformers including an extended all trans and several low energy conformers with one or more gauche segments. This creates a twisting of the chain and facilitates a spatial communication between the alkyl chain and the glycerol backbone as well as between the alkyl chain and the headgroup. The methylene groups of the thioglycerol backbone and the headgroup are the least mobile while the methine group of the thioglycerol backbone appears to have an intermediate mobility. The conformation of the thioether lipid in the two media may be of relevance during its interaction with its site of action, the cellular membrane. Such a conformation may also play an important role in determining the selectivity of this interaction with different cell membranes.

Studies on the thermotropic effects of cannabinoids on phosphatidylcholine bilayers using differential scanning calorimetry and small angle X-ray diffraction.

We have studied the thermotropic properties of a wide variety of cannabinoids in DPPC bilayers. The molecules under study were divided into four classes: (a) classical cannabinoids possessing a phenolic hydroxyl group; (b) delta9-THC metabolites with an additional hydroxyl group on the C ring; (c) non-classical cannabinoids, and (d) cannabinoids with a protected phenolic hydroxyl group. The results showed that the first three groups have similar effects on the thermotropic properties of DPPC bilayers up to x = 0.05 (molar ratio) and that these effects do not parallel their biological activity. For concentrations less than x = 0.01, cannabinoids affect mainly the pretransition temperature in a progressive manner until its final abolishment. At x = 0.05, they further affect the main phase transition by lowering its phase transition temperature and broadening its half width. At high concentrations the thermograms have multiple components, indicating that membranes are no longer homogeneous but rather consist of different domains. At these concentrations cannabinoids with more hydroxyl groups give simpler thermograms. Low concentrations of cannabinoids in group d affect significantly the pretransition temperature, while high concentrations affect only marginally the main phase transition by slightly lowering its temperature and broadening its half width. These results point out the importance of the phenolic hydroxyl group in inducing membrane perturbations. The d-spacing data from our small angle X-ray diffraction experiments show that delta8-THC produces significant structural changes in the lipid bilayer, including the gel-phase tilting angle, the intermolecular cooperativity and the gauche:trans conformer ratio. Conversely, the inactive analog Me-delta8-THC does not cause drastic changes to the bilayer structure.

Topography and thermotropic properties of cannabinoids in brain sphingomyelin bilayers.

In our previous publications we compared the locations of the biologically active (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of its inactive analog O-methyl-(-)-delta 8-tetrahydrocannabinol (Me-delta 8-THC) in the liquid crystalline phase of partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers (Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was shown to localize itself preferentially in the vicinity of the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper towards the center of the bilayer. In the present publication we studied and compared the topography of the two analogs in the gel phase of brain sphingomyelin bilayers. Again we found that delta 8-THC is located near the membrane interface approximately 15 A from the center of the bilayer while its inactive analog localizes deeper in the bilayer at an average site only 8 A from the center of the membrane bilayer. It thus, appears that both analogs preferentially localize in distinct sites within the membrane bilayer which are independent of the mesomorphic state and the nature of the phospholipid. Our results suggest that in the more complex environment of biological membrane which is composed of different phospholipids and proteins the two analogs are expected to prefer different average locations within the bilayer, a property which may in part explain the observed differences in their biological activities.

Effects of the anesthetic steroid alphaxalone and its inactive delta 16-analog on the thermotropic properties of membrane bilayers. A model for membrane perturbation.

We have studied in detail the effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on the thermotropic properties of model membranes using differential scanning calorimetry (DSC). The results obtained showed that, for model membranes from hydrated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and egg sphingomyelin, the biologically active analog significantly broadened the phase transition, in contrast to the inactive one which produced only marginal effects. Also, alphaxalone abolished the pretransition in these preparations whereas its delta 16-analog only broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results suggest that the ability of the two steroids to perturb membranes is lipid dependent. Comparisons between the effects of the two steroids on lipid/cholesterol model membranes revealed that delta 16-alphaxalone excluded cholesterol from lipid/cholesterol/delta 16-alphaxalone ternary systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol system. In an attempt to determine whether the different thermotropic effects of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of incorporation into the bilayer, solid state 2H-NMR was applied using specifically deuterated steroids. The 2H-NMR data showed that alphaxalone incorporated fully into the membrane bilayer up to a molar concentration of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membrane preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bilayers were studied using DSC. The experiment showed that alphaxalone perturbed the membrane bilayers more effectively than its inactive analog. These results strongly suggest that the small structural differences between the two steroids are responsible for the observed differences in their abilities to perturb membranes, possibly because of differences in the packing of these two molecules within the bilayers.